Abstract
Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.
Highlights
Cereal grains can be stored for a year or more following harvest before they are milled for food and feed, during which time they are subject to infestation by various species of stored-product pests [1]
This work confirms that point mutations in the dihydrolipoamide dehydrogenase (DLD) gene are associated with strong resistance to phosphine in the beetle species T. castaneum and R. dominica, that these resistance genes occur in U.S populations of these two species, increasing the geographic extent of this resistance from Australia [14] and India [20]
For T. castaneum we found only a single resistance SNP for an amino acid change of P to S (P45S) that was unknown in Australia though common in India, while the only isolated Australian resistance allele for T. castaneum [14] was for a G to S (G131S) SNP at a different position in the DLD gene
Summary
Cereal grains can be stored for a year or more following harvest before they are milled for food and feed, during which time they are subject to infestation by various species of stored-product pests [1]. Opit et al [8] used laboratory susceptible populations of these pest species to compute resistance ratios for the toxicity of phosphine for resistant populations compared to susceptible populations, based on the estimated lethal concentration to kill 99% of the sampled population (the LC99) following dose-mortality experiments. They found that the most resistant T. castaneum population required a phosphine dose for control that was 119-fold greater than that needed for susceptible beetles, and that the most resistant R. dominica population required a phosphine dose that was 1520-fold higher than that for susceptible insects
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